The structure of XK in the apo form has been determined at 2.7 Å resolution using MIR phasing. The apo structure was used to phase a xylulose-bound structure at 2.1 Å. This model consists of two protein molecules (A and B) that form the asymmetric unit, each consisting of residues 1-334 and 343-484 of the 484 predicted. There was no electron density corresponding to the region from 335 to 342. There are also 310 water molecules which are observed in the asymmetric unit. The average temperature factor is 37.9 Å2
for molecule A and 44.2 Å2
for molecule B. Modeling and more detailed structural analysis therefore focused on molecule A. The r.m.s.d. between the α-carbons of each monomer is 0.95 Å with no major differences. A Ramachandran plot generated by PROCHECK indicates 90.3% of non-glycine residues in the core regions, 9.0% are in additionally allowed and 0.7% are in generously allowed areas 18
. Each monomer of XK is 76 × 46 × 56 Å and is composed of two major domains (I and II) connected by a hinge segment composed of residues Ala294 to Asp299 (α-helix α11) (). Positive Fo
density was found in the active site on both monomers which was interpreted as a xylulose molecule. Statistics associated with the final refinement are shown in .
Figure 1 The main chain trace of XK. A) A stereo view of XK looking into the substrate binding cleft. The molecule is colored from red (domain IA), pink (domain IB) to blue (domain IIA) and light blue (domain IIB). The hinge segment is colored yellow. B) A stereo (more ...)
Data collection and refinement statistics. Numbers in parentheses describe values for the high resolution shell. EMTS: ethylmercurythiosalicylate
Using a VAST search to screen the Protein Data Bank (PDB), the ecGK structure was found to be the most homologous structure but also indicated XK shares structural features with the other members of the ATPase superfamily 19
. The two enzymes have only 20% sequence identity, but an overlap of the domains individually indicates they are structurally homologous. An r.m.s.d. of 1.6 Å was calculated between 264 residues in domain I and 2.0 Å for 153 residues in domain II. Other structures including 3-phosphoglycerate kinase, hexokinase and NAD kinase also share the same overall ATPase scaffold with two domains linked with a hinge region 20; 21; 22
. As described previously for the ecGK structure, domain I is primarily responsible for sugar substrate binding and domain II is mainly involved in ATP binding 23
. Substrates bind deeply in the cleft formed between these two domains. These domains can be further divided into subdomains where IA and IIA form the ATPase core () of XK and subdomain IB forms part of the D-xylulose binding site. Subdomain IIB is involved in the intermolecular interface of the XK dimer, mediated by β-sheets β18 and β19 (, ). As observed in ecGK subdomain IA contains the conserved catalytic residues and is responsible for the phospho-group transfer function.
Figure 2 Oligomeric structure of XK. A) The dimer observed within the asymmetric unit of XK resembles the O/Y dimer found in the allosterically-inhibited ecGK tetramer. A zoomed view of XK dimeric interface is shown and illustrates the interactions between β-interactions (more ...)
Regulation of carbohydrate kinase members
In ecGK, fructose 1,6-bisphosphate (FBP) inhibits the enzyme by creating an inactive tetramer from two active dimers by mediating interactions between an extended loop from each domain I across the O/X and Y/Z interfaces () 15
. The binding site is primarily formed by Gly233, Gly234 and Arg236 located on each side. XK has no known allosteric regulators and a comparison shows that the O/X or Y/Z interfaces are different between ecGK and a hypothetical XK tetramer 14
. In XK, the corresponding region is structurally different from GK with a short α-helix substituted for the loop ( and ). Also none of the residues involved in the tetramer formation in GK are found in XK and especially the Arg236 (Thr224 in XK) described to be important for the tetramerization 15
Figure 6 Pairwise sequence alignment of XK and ecGK. Green designates residues involved in tetramer formation in ecGK; yellow, the XK hinge segment; red, the dimeric interface of both XK end ecGK. Residues composing each XK domain and subdomain are as follows: (more ...)
The two molecules in the crystallographic asymmetric unit do clearly maintain the dimeric contact found between the O/Y and X/Z ecGK dimers which are present along the vertical axes in 15
. The XK dimeric interface is primarily composed of an antiparallel β-sheet contact composed of the segment from 345 to 359 ( and ). This is structurally homologous with what is found in ecGK subdomain IIB. Residues composing the β-sheet and mediating the interface are 80% identical between ecGK and XK. This is also a well-conserved region across the kinase family implying other members are dimeric as well.
There are alternate modes of regulation found in other sugar kinase members, particularly GK. Phosphorylation of His232 in En. casseliflavus GK, which is present in a loop mediating the O/X interface (), has been found to activate the enzyme. The phosphotransferase system is known to allosterically regulate ecGK via interactions with the phosphocarrier protein IIAGlc. Xylose and xylulose are not recognized by this system and the IIAGlc binding site on the carboxyl terminus of GK is not conserved in sequence or structure in XK.
Since XK appeared to be a tenuously associated dimer in the crystal structure, cryo electron microscopy with 3D map reconstruction were used to confirm this arrangement in solution. shows projection structures of XK in different orientations. The low molecular weight of protein made particle selection difficult, but the class averages in clearly document the dimeric nature of XK. In order to increase the signal to noise ratio, class averages were calculated and used for the 3D reconstruction, (). The final reconstruction was resolution limited to 36 Å resolution ( and ). The docking of the XK dimer () into the 3D density map strongly supports the dimeric arrangement observed in the crystal structure. A real space correlation coefficient of 85.5% was calculated by overlapping 36 Å maps and indicates a high level of agreement. The XK dimeric state was further confirmed by dynamic light scattering experiment which shows a molecular weight of 110 kDa (data not shown).
Conservation of the XK ATPase Core Structure
Subdomains IA and IIA form the ATPase core and they each contain a five stranded β-sheet flanked by three α-helices (). This architecture has previously been observed in other superfamily members such as ecGK, yeast hexokinase, actin and the DNAse domains of hsc70 and DnaK 12; 13; 24; 25
. Catalytic residues are also well conserved among the superfamily including two conserved aspartate, glutamate, or glutamine residues at the active site which have key roles in the catalytic mechanism (). The first of these functions as a general base, assisting the removal of a proton from the attacking hydroxyl group (Asp233 in XK). The second is an aspartate residue in domain I which interacts with the Mg2+
ion complexed with the nucleotide (Asp6 in XK). The positions of the aspartates are very similar when XK is overlayed with structures of the rabbit actin (PDB accession number 1QZ5), the yeast apo-hexokinase PII (1IG8), the E. coli
DnaK (1DKG) or ecGK (1GLF). Residues expected to be responsible for specificity are spatially divergent and none of the residues involved in the xylulose binding in XK are found in the human hexokinase type 1 complexed with D-glucose (1HKC).
Multiple ATPase sequence alignment with catalytic residues shown in red.
Apparent kinetic parameters of XK for the phosphorylation of D-xylulose, D-ribulose, D-arabitol and xylitol are summarized in . In the absence of substrate, XK has a weak but significant ATPase activity for which kinetic parameters are also shown. This ATPase activity, recorded at a fixed MgATP concentration of 5 mM, was strongly (13-fold) inhibited in the presence of the non-phosphorolyzable inhibitor 5-F-xylulose (0.18 mM), suggesting that hydrolysis of ATP in the absence of substrate is an inherent property of XK and does not result because of contamination of the enzyme preparation with another ATPase. The results in reveal a relatively relaxed substrate specificity of the enzyme. However, in kcat/Km terms D-xylulose is either preferred or strongly preferred over the other substrates tested. Up to 1300-fold changes in catalytic efficiency are observed in response to alterations of substrate structure () and reflect mainly increases in the apparent Michaelis constant. The value of kcat, in contrast, was almost constant across the substrate series, suggesting a common rate-determining step in the reaction of XK with the different compounds. The marked 50-fold decrease in kcat/Km caused by inversion of C3 chirality in D-xylulose to D-ribulose shows the high stereochemical selectivity of XK (). Replacement of the C2 carbonyl group of D-xylulose by an L-configured hydroxyl group in xylitol brought about an even larger 460-fold decrease in catalytic efficiency. In addition, it caused a complete loss of synergism in the apparent binding of substrate and MgATP. Using the apparent Michaelis constant for the ATPase reaction of XK as reference, MgATP was 41-fold more tightly bound when D-xylulose was present at a saturating concentration. The model of the XK ternary complex provides explanations for these kinetically determined effects, showing a sugar-dependent domain closure.
Table 3 Apparent kinetic parameters for several XK substrates. Constants were calculated by fit of equation 1 to initial rate data.
Sugar substrates used in substrate specificity studies
A significant level of synergism was also observed in the binding of the dead-end inhibitor 5-F-xylulose and MgATP. The 5-F-xylulose is a linear competitive inhibitor with respect to D-xylulose. The value of Kic for the inhibitor decreased ≈ 6-fold in response to a 30-fold increase in the fixed concentration of MgATP from the KmATP level to a saturating level (). Interestingly, binding of D-xylulose and AMPPNP, a linear competitive inhibitor with respect to MgATP, was not synergistic (), suggesting that the dead-end inhibitor differs from MgATP in properties essential for the ligand-induced crosstalk of the XK domains. The substrate selectivities and the resulting synergistic binding of ATP are explained by results of modeling experiments in which the ternary complex was modeled with both ATP and xylulose using the structure of the ecGK ternary complex as the basis (described below).
Inhibition patterns and constants from inhibition studies with dead-end inhibitors.
Kinetic Mechanism of XK from Initial Rate and Dead End Inhibition Patterns
The steady-state kinetic mechanism of XK was examined by determining initial rate patterns. A double reciprocal plot of initial rates recorded at varied concentrations of D-xylulose at several constant concentrations of MgATP shows an intersecting pattern () implying that D-xylulose and MgATP must bind to the enzyme before the first product is released. Dead-end inhibitor binding studies were used to determine the order of substrate addition in the sequential kinetic mechanism of XK (). As described above, 5-F-xylulose and AMPPNP are competitive inhibitors with respect to D-xylulose and ATP respectively. AMPPNP is a linear uncompetitive inhibitor with respect to D-xylulose concentration, measured at Km
levels of ATP (). The presence of 5-F-xylulose induced substrate inhibition by MgATP under conditions in which initial rates were measured at a varied concentration of MgATP and a constant Km
concentration of D-xylulose, as shown in . These results suggest a kinetic mechanism of XK. The presence of ATPase activity in the absence of substrate indicates that the mechanism is probably formally random but a path in which substrate binds before MgATP is strongly preferred. In a predominantly ordered mechanism, binding of a dead-end inhibitor (I = AMPPNP) that is competitive with respect to the second substrate (B = MgATP) is expected to give uncompetitive inhibition with respect to the first substrate (A = D-xylulose). The proposed mechanism explains substrate inhibition by MgATP induced by 5-F-xylulose due to formation of an abortive E-I-B (I= 5-F-xylulose) complex that prevents release of the inhibitor, as shown in . In a fully random mechanism, substrate inhibition by B is not observed under the conditions in because the inhibitor readily dissociates from the E-I-B complex, resulting in E-B, which is free to combine with A to yield products. The results reveal that XK shares kinetic properties with E. coli
and yeast hexokinase 27
. ecGK was reported to have a random kinetic mechanism 28
and ribulokinase from Klebsiella aerogenes
appears to use a steady-state random mechanism 29
Figure 4 Kinetic results and proposed kinetic mechanism. A) Double-reciprocal plot of initial rate data for XK with D-xylulose varied at MgATP concentrations of 0.05 mM, squares; 0.13 mM, circles; 0.49 mM, triangles up; 1.46 mM, diamonds; 2.44 mM, hexagons; 4.88 (more ...)
Initial rates shown in were fitted to eq. 2
and kinetic parameters are summarized in . The apparent dissociation constants for the binary complexes of XK with D-xylulose (KiA
) and 5-F-xylulose (Kic
determined at a non-saturating MgATP level) are similar, indicating that the fluorine can substitute well for the reactive hydroxy group in the initial substrate binding recognition. This is an important result in view of the proposed mechanistic scenario for XK and related members of this family of carbohydrate kinases (see below for details) probably involves activation of the 5-OH through proton abstraction by a catalytic base. The fluorine obviously could not replace the original hydroxyl group if binary complex interactions involved a hydrogen bond from the 5-OH to the base. The findings suggest that formation of the ternary complex is required to correctly align the catalytic groups on the enzyme and the reactive parts of the substrates.
Table 5 Kinetic constants for E. coli xylulokinase at pH 7.4 from fit of equation 2 to initial rate data.
The predominantly ordered mechanism of XK implies that the value of kcat/Km essentially reflects the bimolecular rate constant of substrate binding (kon) to the free enzyme. kon therefore governs the sugar substrate selectivity (), probably through a conformational mechanism. The relative constancy of kcat under conditions when kon changes more than 5000-fold suggests that ternary complex interconversion is presumably the rate-determining step of the reaction ( and ).
Conformational Changes and Substrate Binding
The subject of substrate-induced conformational changes in sugar kinases has been implied by the observed cooperative binding of substrates in many of them and the observation that there is restricted solvent accessibility in structures of bound substrates. Domain motions on the order of only 7º have been observed when comparing wild-type and mutant GK14
which are different from the 12º movement observed for the more distantly related hexokinase 30
. The apo and xylulose-bound structures coupled with modelling indicate that the domain closure in XK is dramatically greater than movements seen in closely related carbohydrate kinases before. Overlaying the N-terminal domains of the unbound and xylulose-bound XK shows that xylulose binding induces a 12.2º closure which increases when ATP binds in modelling experiments (see below). The second molecule in the asymmetric unit of the apoenzyme structure is actually closed 1.0º relative to the xylulose-bound form indicating that there is conformational flexibility. Similar flexibility is also found in the case of human glucokinase which exists in three different forms: super-open, open and closed 31
. The enzyme undergoes a conformational change from the super-open to open-form when glucose binds and then to closed-form in the presence of ATP. Assuming that the mechanism of synergistic binding described for human glucokinase can be applied to XK, the apo form may represent the super-open form and the xylulose-soaked structure corresponds to the open form.
A comparison of the apo and xylulose-bound forms of XK shows that the helical segment 294-299 (in α11) in domain IIA (, and ) acts as a hinge. Although there are no significant structural effects of hinge movement on domain I (Cα r.m.s. deviation between apo and xylulose bound structures is only 0.75Å), effects are seen on domain II (rmsd of 3.7 Å). The movements in domain II involve a rigid-body motion toward the domain I along with a slight 5º yaw across an axis between the N-term to C-term of the structure (). The domain motion also induces a closure at the bottom of the cleft by bending the four parallel β-strands of domain IIA which are part of the interface between domain IA and IIA (, ).
Figure 5 Substrate-induced conformational changes in XK and modeling of the ternary structure using ecGK. A) A stereo view of domains I overlaps from apo XK (blue), xylulose bound XK (green) and the ecGK ternary complex (red) shows a ~37° opening of the (more ...)
To determine conformational changes associated with ternary complex formation, XK crystals were soaked with xylulose and ATP-γS but rapidly dissolved, probably reflecting a change disrupting the lattice. Modeling was done to close xylulose-bound XK using the closed, ternary complex of ecGK as a template. This yielded a model in which the domains are now 37º closed relative to the apo form. Hydrogen bonding interactions mediating xylulose binding come from the sidechains of His78, Asp233, Asn234 and the Met77 backbone nitrogen, all belonging to the domain IA. Xylulose bridges the cleft by interacting with a mobile Ser256 which resides on a loop via a hydrogen bond from domain IIA. Non-polar interactions involving the side chain of Trp96 also contribute to xylulose binding in the active site. Compared to xylulose, xylitol is a much poorer substrate and is not synergistic with ATP binding (). Modeling indicates it is only stabilized by 2 hydrogen bonds (Thr9 and backbone nitrogen from Met77) and polar interactions with Trp96 and Asp233. It does not seem to bridge the cleft by interacting with Ser256 or any residues on the flexible loop from domain IIA as observed for the xylulose binding
The catalytic residues (Asp6, Thr9 and Asp233) are conserved across the ATPase superfamily including ecGK (, ) but the residues in the triphosphate binding site are more variable and none of the ATP binding residues are conserved in XK 23
. Modeling ATP binding is therefore more difficult but plausible hydrogen bonds Asp6, Thr9 and Asp233 in domain IA and Thr255, Asp299 and Pro311 in domain IIA may bridge the cleft (). In the model of the closed ternary complex, 4% of the 284 Å2
surface of the sugar is solvent accessible and this increases to 14% when the ATP molecule is removed, consistent with the kinetically ordered mechanism.
Catalytic Mechanism of XK
Structural overlays indicate Asp6 and Asp233 of XK are homologous in their positions to Asp10 and Asp245 of ecGK 23
. A crucial role of Asp6 and Asp233 in the XK catalytic mechanism can be inferred from biochemical studies of relevant mutants (D10N, D245N) of ecGK as well as from the conservation pattern of the two residues in members of the carbohydrate kinase family 32
. Analysis of kinetic consequences in D6A and D233A variants confirmed that both Asp residues are essential for XK activity. Enzymatic rates of phosphorylation of D-xylulose were at the limit of detection, ~4.5 orders of magnitude below the level of wild-type activity. The substrate-independent ATPase activity seen in the wild-type (), however, was much less affected than the phospho-group transfer activity in D233A mutant (kcat
= 0.13 ± 0.01 s−1
= 8.2 ± 1.2 mM) which displays a ~2-fold increase in the original value of kcat
for this activity. The ATPase activity of the D6A mutant (kcat
≈ 0.0038 s−1
), by contrast, was significantly (19-fold) decreased, compared with the wild-type activity.
Results of structural analysis, modeling and site-directed mutagenesis suggest a mechanism for XK which is consistent with mechanisms for other family members such as ecGK but also reveals new details (). A conserved role of the side chain of Asp6 in coordinating and positioning the MgATP for catalysis, is supported for XK. An additional function served by Asp6 (in concert with Mg2+ ligated by it) might be the stabilization of the ADP leaving group during phospho-group transfer to sugar or water.
The proposed catalytic mechanism for XK.
The role of Asp233 is to deprotonate the D-xylulose hydroxyl at the 5 position, activating it for nucleophilic attack on the phosphate. The main chain NH group from Thr9 could stabilize the transition state and the associated negative charge development on O5 through a hydrogen bond (). The relative timing of bond forming and bond cleaving steps in the phospho-group transfer catalyzed by kinases and hydrolases as well as the question of whether the transition state has a rather dissociative or associative character have attracted much attention among enzymologists and is still under debate. The extent to which base catalysis contributes to stabilization of the transition state depends on the nature of bonding in it. Bond cleavage in the leaving group at little or no bond formation in the incoming nucleophile dominates in the dissociative transition state which is characterized by a decrease in the combined bond order to the incoming and departing groups. In that extreme case, base catalysis to the removal of a proton would provide no significant advantage. The 104.5-fold loss of activity in D233A and the charge-stabilizing hydrogen bond from the backbone amide of Thr9 would suggest a significant nucleophilic participation of the 5-OH in the transition state. The replacement of the putative catalytic base in related kinases showed a range of effects, but typically ≥ 102.7-fold losses of activity irrespective of the character of the residue introduced by the mutation. Although it is possible that Asp233 and its homologues in other kinases are involved in the correct positioning of the reactive hydroxyl group for nucleophilic attack, the magnitude of the functional disruption caused in the mutants suggests direct participation in catalysis, as a base, to be the major role of them.